Bone sialoprotein (BSP) is one of the major non-collagenous extracellular matrix (ECM) phosphoproteins in bone and dentin. BSP is highly expressed in developing and postnatal regenerating bone, but its precise function is largely unclear. Previous in vitro studies suggest that BSP may be involved in cell attachment, cell migration and calcium-phosphate mineral deposition. We have recently investigated the function of BSP in more complex in vivo environments by implanting BSP with type-I collagen as a carrier into surgically created 8-mm rat cranial defects and thoracic subcutaneous pouches. The results show that BSP-collagen, but not collagen alone, stimulates differentiation of resident responding cells into osteoblasts, followed by synthesis of new bone in the mid-portion of the cranial defects. These responding cells are primarily derived from the dura mater. DNA microarray and quantitative real-time PCR analyses reveal that the expression of specific members of the Wnt and bone morphogenetic protein (BMP) families are up-regulated in the repair cells within cranial BSP-collagen implants compared to those in cranial collagen implants. In contrast, no osteogenesis is observed when BSP-collagen is implanted into rat thoracic subcutaneous tissues. The bioactivity of BSP in stimulating osteoblast differentiation and bone regeneration in a bone defect model is an unprecedented result; however, the mechanisms of BSP-mediated osteogenesis are unknown. In this application we wish to explore the mechanisms for BSP-specific osteogenic action through three specific aims. Specific Aim 1 will investigate the mechanisms for tissue-specific response to BSP treatment. Specific Aim 2 will identify key signaling pathways through which BSP stimulates osteoblast differentiation and its ensuing osteogenesis. Specific Aim 3 will identify BSP-specific osteogenic activities for the repair of cranial defects.